Project Summary X-Linked Intellectual Disability (XLID) affects approximately 1 in 500 males in the United States. We have identified several mutations in the O-GlcNAc Transferase gene (OGT) that are causal for XLID, but the mechanism underlying the phenotype is unknown. OGT is an essential nucleocytoplasmic glycosyltransferase that modifies nuclear and cytosolic proteins with a single ?-N-Acetyl-Glucosamine (O-GlcNAc). OGT has thousands of substrates and O-GlcNAc serves a diverse set of functions, including modulating nutrient sensing, transcription, and synaptic function. The O-GlcNAc modification is considered analogous to phosphorylation, but unlike kinases, OGT is the only enzyme responsible for the O-GlcNAc modification within the mammalian cell. Therefore, the mechanism of OGT substrate selectivity is a major area of interest. It is thought that the N-terminal tetratricopeptide repeats (TPRs) of OGT are responsible for OGT substrate selection in part by recruitment of partner proteins that target OGT to specific substrates and cellular domains. All of the OGT XLID variants being studied here are localized to the TPRs, leading to our hypothesis on the mechanism by which OGT mutations lead to XLID: that rather than interrupting the stability or catalytic activity of OGT, the OGT XLID variants exhibit impaired protein-protein interactions and that these anomalous protein interactions cause disruptions in cellular function that lead to the XLID phenotype. This hypothesis is supported by data demonstrating that all of the OGT XLID variants are thermally stable, catalytically functional, and kinetically comparable to the wild-type (WT) OGT. To test our hypothesis, we will use an unbiased proximity proteomic approach to define the WT and XLID OGT interactomes, and identify the cellular impact of aberrant interactions. We are uniquely poised to address this hypothesis due to our expertise in O-GlcNAc biology, mass spectrometry, and our possession of Cas9- engineered male human embryonic stem cells expressing each OGT XLID variant. In Aim 1, we will use a proximity proteomic method, BioID, to identify OGT TPR interactors. WT OGT and XLID variant interactomes will be compared to identify aberrant interactions and individual interactions validated. In Aim 2, we will assess the molecular contribution of aberrant interactions in the XLID phenotype, using a variety of assays to assess the interactor?s characteristics (localization, expression, post-translational modifications) and functional consequences of its loss of interaction with OGT (enzymatic, transcriptomic, signaling pathway analyses). These approaches will not only define a model for how OGT TPR mutations cause XLID, but also identify a WT OGT TPR interactome, an essential resource for the field. This research will take place in Dr. Lance Wells? lab at the University of Georgia, placing the trainee in an excellent environment to learn general and specialized biochemical skills under outstanding mentorship. The training plan, while focused on developing the trainee into an independent scientist, also integrates DVM clinical training and research development to ensure the trainee gains the skills necessary to be successful in a career as a veterinary clinician scientist.